Phosphoproteome of the cyanobacterium Synechocystis sp. PCC 6803 and its dynamics during nitrogen starvation.

Spät P, Maček B, Forchhammer K - Front Microbiol (2015)

Bottom Line:
PCC 6803.Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism.This study reveals dynamics of Synechocystis phosphoproteome in response to environmental stimuli and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of homeostatic control in cyanobacteria.

ABSTRACTCyanobacteria have shaped the earth's biosphere as the first oxygenic photoautotrophs and still play an important role in many ecosystems. The ability to adapt to changing environmental conditions is an essential characteristic in order to ensure survival. To this end, numerous studies have shown that bacteria use protein post-translational modifications such as Ser/Thr/Tyr phosphorylation in cell signaling, adaptation, and regulation. Nevertheless, our knowledge of cyanobacterial phosphoproteomes and their dynamic response to environmental stimuli is relatively limited. In this study, we applied gel-free methods and high accuracy mass spectrometry toward the detection of Ser/Thr/Tyr phosphorylation events in the model cyanobacterium Synechocystis sp. PCC 6803. We could identify over 300 phosphorylation events in cultures grown on nitrate as exclusive nitrogen source. Chemical dimethylation labeling was applied to investigate proteome and phosphoproteome dynamics during nitrogen starvation. Our dataset describes the most comprehensive (phospho)proteome of Synechocystis to date, identifying 2382 proteins and 183 phosphorylation events and quantifying 2111 proteins and 148 phosphorylation events during nitrogen starvation. Global protein phosphorylation levels were increased in response to nitrogen depletion after 24 h. Among the proteins with increased phosphorylation, the PII signaling protein showed the highest fold-change, serving as positive control. Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism. This study reveals dynamics of Synechocystis phosphoproteome in response to environmental stimuli and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of homeostatic control in cyanobacteria.

Figure 2: Immunoblot validation of PII protein and phosphorylation dynamics. The change in protein abundance (SDS PAGE) and phosphorylation (Clear native PAGE) of the N-regulatory PII protein between nitrate- (NO−3) or ammonia-grown (NH+4) and 24h h nitrogen starved cells (-N24h) from both experiments is shown on the left panel. P0II represents the unphosphorylated trimeric complex and P1–3II represent P II trimers with one, two and three phosphorylated subunits. The AtpB protein serves as loading control for SDS and Clear native PAGE. Corresponding MS acquired protein and phosphorylation event ratios in log2 scale, relative to the NO−3 are shown on the right panel.

Mentions:
As a test case for our quantitative mass spectrometry acquired (phospho)proteome data, we analyzed the expression profile of the nitrogen regulatory signal protein PII by immunoblot analysis on the protein level by SDS-polyacrylamide gel electrophoresis (PAGE) and on the phosphorylation level by non-denaturing PAGE (Forchhammer and Tandeau De Marsac, 1994). Under nitrogen-limiting conditions, the PII signal protein accumulates, associated with enhanced phosphorylation of residue Ser49 at the apex of the surface exposed T-loop (Forchhammer, 2004). Under nitrogen-depleted conditions, all three subunits of the homotrimeric PII protein are phosphorylated (Forchhammer and Tandeau De Marsac, 1994). In the present experiments, PII protein levels were similar between nitrate or ammonium grown cells but increased under nitrogen starvation (Figure 2). This is in accordance with transcription analysis of the glnB gene (encoding the PII protein), showing up-regulation under nitrogen starvation (Fadi Aldehni et al., 2003; Aguirre Von Wobeser et al., 2011). The result of the analysis of the PII phosphorylation state by non-denaturing PAGE from both experiments is shown below in the figure. As expected, the Ser49 phosphorylation strongly increased under nitrogen starvation, with the phosphorylated PII trimer isoforms becoming the most prominent bands. The unphosphorylated PII protein is in contrast the most intense band in nitrate and ammonia grown conditions. Overall, MS based quantification of the PII signaling protein with respect to protein abundance as well as on the phosphorylation level, as indicated in the figure, is in good agreement with the immunoblot results, implying that the quantitative MS analysis is valid.

Figure 2: Immunoblot validation of PII protein and phosphorylation dynamics. The change in protein abundance (SDS PAGE) and phosphorylation (Clear native PAGE) of the N-regulatory PII protein between nitrate- (NO−3) or ammonia-grown (NH+4) and 24h h nitrogen starved cells (-N24h) from both experiments is shown on the left panel. P0II represents the unphosphorylated trimeric complex and P1–3II represent P II trimers with one, two and three phosphorylated subunits. The AtpB protein serves as loading control for SDS and Clear native PAGE. Corresponding MS acquired protein and phosphorylation event ratios in log2 scale, relative to the NO−3 are shown on the right panel.

Mentions:
As a test case for our quantitative mass spectrometry acquired (phospho)proteome data, we analyzed the expression profile of the nitrogen regulatory signal protein PII by immunoblot analysis on the protein level by SDS-polyacrylamide gel electrophoresis (PAGE) and on the phosphorylation level by non-denaturing PAGE (Forchhammer and Tandeau De Marsac, 1994). Under nitrogen-limiting conditions, the PII signal protein accumulates, associated with enhanced phosphorylation of residue Ser49 at the apex of the surface exposed T-loop (Forchhammer, 2004). Under nitrogen-depleted conditions, all three subunits of the homotrimeric PII protein are phosphorylated (Forchhammer and Tandeau De Marsac, 1994). In the present experiments, PII protein levels were similar between nitrate or ammonium grown cells but increased under nitrogen starvation (Figure 2). This is in accordance with transcription analysis of the glnB gene (encoding the PII protein), showing up-regulation under nitrogen starvation (Fadi Aldehni et al., 2003; Aguirre Von Wobeser et al., 2011). The result of the analysis of the PII phosphorylation state by non-denaturing PAGE from both experiments is shown below in the figure. As expected, the Ser49 phosphorylation strongly increased under nitrogen starvation, with the phosphorylated PII trimer isoforms becoming the most prominent bands. The unphosphorylated PII protein is in contrast the most intense band in nitrate and ammonia grown conditions. Overall, MS based quantification of the PII signaling protein with respect to protein abundance as well as on the phosphorylation level, as indicated in the figure, is in good agreement with the immunoblot results, implying that the quantitative MS analysis is valid.

Bottom Line:
PCC 6803.Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism.This study reveals dynamics of Synechocystis phosphoproteome in response to environmental stimuli and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of homeostatic control in cyanobacteria.

ABSTRACTCyanobacteria have shaped the earth's biosphere as the first oxygenic photoautotrophs and still play an important role in many ecosystems. The ability to adapt to changing environmental conditions is an essential characteristic in order to ensure survival. To this end, numerous studies have shown that bacteria use protein post-translational modifications such as Ser/Thr/Tyr phosphorylation in cell signaling, adaptation, and regulation. Nevertheless, our knowledge of cyanobacterial phosphoproteomes and their dynamic response to environmental stimuli is relatively limited. In this study, we applied gel-free methods and high accuracy mass spectrometry toward the detection of Ser/Thr/Tyr phosphorylation events in the model cyanobacterium Synechocystis sp. PCC 6803. We could identify over 300 phosphorylation events in cultures grown on nitrate as exclusive nitrogen source. Chemical dimethylation labeling was applied to investigate proteome and phosphoproteome dynamics during nitrogen starvation. Our dataset describes the most comprehensive (phospho)proteome of Synechocystis to date, identifying 2382 proteins and 183 phosphorylation events and quantifying 2111 proteins and 148 phosphorylation events during nitrogen starvation. Global protein phosphorylation levels were increased in response to nitrogen depletion after 24 h. Among the proteins with increased phosphorylation, the PII signaling protein showed the highest fold-change, serving as positive control. Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism. This study reveals dynamics of Synechocystis phosphoproteome in response to environmental stimuli and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of homeostatic control in cyanobacteria.